Cross-species Sequence Analysis Reveals Multiple Charged Residue-rich Domains That Regulate Nuclear/Cytoplasmic Partitioning and Membrane Localization of A Kinase Anchoring Protein 12 (SSeCKS/Gravin)

Abstract

A kinase anchoring proteins (AKAPs) assemble and compartmentalize multiprotein signaling complexes at discrete subcellular locales and thus confer specificity to transduction cascades using ubiquitous signaling enzymes, such as protein kinase A. Intrinsic targeting domains in each AKAP determine the subcellular localization of these complexes and, along with protein-protein interaction domains, form the core of AKAP function. As a foundational step toward elucidating the relationship between location and function, we have used cross-species sequence analysis and deletion mapping to facilitate the identification of the targeting determinants of AKAP12 (also known as SSeCKS or Gravin). Three charged residue-rich regions were identified that regulate two aspects of AKAP12 localization, nuclear/cytoplasmic partitioning and perinuclear/cell periphery targeting. Using deletion mapping and green fluorescent protein chimeras, we uncovered a heretofore unrecognized nuclear localization potential. Five nuclear localization signals, including a novel class of this type of signal termed X2-NLS, are found in the central region of AKAP12 and are important for nuclear targeting. However, this nuclear localization is suppressed by the negatively charged C terminus that mediates nuclear exclusion. In this condition, the distribution of AKAP12 is regulated by an N-terminal targeting domain that simultaneously directs perinuclear and peripheral AKAP12 localization. Three basic residue-rich regions in the Nterminal targeting region have similarity to the MARCKS proteins and were found to control AKAP12 localization to ganglioside-rich regions at the cell periphery. Our data suggest that AKAP12 localization is regulated by a hierarchy of targeting domains and that the localization of AKAP12-assembled signaling complexes may be dynamically regulated.Multicellular organisms require extensive signaling networks to coordinate physiological responses to a diverse array of stimuli. As organismal complexity increases, so theoretically does the requirement for an increased complement of signal transduction machinery. Whereas expansion of some kinase families appears to parallel increasing organismal complexity (1), the protein kinase A family has undergone little expansion (2) despite the large number of its physiological substrates distributed throughout the cell. Thus, intrinsic differences between protein kinase A family members cannot fully account for such diversity because, on their own, they do not provide sufficient specificity. Instead, specificity appears to be conferred by an ever expanding pseudo-family of anchoring proteins, the A kinase anchoring proteins (AKAPs), 1 that compartmentalize protein kinase A-containing signaling complexes to discrete subcellular locales (3).The intrinsic targeting and protein-protein interaction domains of each AKAP direct correct spatial and temporal assembly of protein kinase A-containing signaling complexes. Such compartmentalization simultaneously shields neighboring complexes from activated s...

Gravin regulates mesodermal cell behavior changes required for axis elongation during zebrafish gastrulation

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“…The most highly conserved domains of Gravin are three short basic regions, which are implicated in reversible electrostatic interactions with the plasma membrane (Fig. 1B;Streb and Miano 2005).…”

Hypoxic induction of AKAP12 variant 2 shifts PKA-mediated protein phosphorylation to enhance migration and metastasis of melanoma cells

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AKAP12α is AssoCiated with Promoter Methylation in Lung Cancer

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